Cellular And Molecular Mechanisms Of Toxin Resistance For Endoplasmic Reticulum Translocating Toxins

The endoplasmic reticulum (ER) is the site of co- and post-translational modification for secretory proteins. In order to prevent vesicular transport and secretion of misfolded or misassembled proteins, a highly regulated mechanism called ER-associated degradation (ERAD) is employed. This pathway recognizes misfolded proteins in the ER lumen and targets them to the cytosol for ubiquitination and subsequent degradation via the 26S proteasome. Sec61 and Derlin-1 are ER pores through which export occurs. AB-type protein toxins such as cholera toxin (CT), Shiga toxin (ST), exotoxin A (ETA), and ricin have evolved means of exploiting the ERAD pathway in order to reach their cytosolic targets. AB-type protein toxins consist of a catalytic A-subunit and a cell-binding B-subunit. The B-subunit recognizes cell surface receptors for the toxin. This begins a series of vesicle trafficking events, collectively termed retrograde trafficking, that lead to the ER. Dissociation of the A and B subunits occurs in the ER, and only the A subunit enters the cytosol. The exact mechanism of A subunit translocation from the ER to the cytosol is unknown. Toxin translocation occurs through a pore in the ER membrane. Exit through the pore requires the toxin to be in an unfolded conformation. The current model for toxin translocation proposes that ER chaperones actively unfold the toxin A chain for translocation. After the translocation event, the toxin spontaneously refolds to an active conformation. Our model suggests that unfolding in the ER is spontaneous and refolding in the cytosol is dependent upon cytosolic chaperones. Based on our model, we hypothesize that blockage of the A subunit unfolding and/or the ERAD translocation step will confer a phenotype of non-harmful multi-toxin resistance to cells. In support of this model, we have shown that, at 37[degrees]C, the isolated catalytic subunit of cholera toxin (CTA1) is in an unfolded and protease sensitive confirmation that identifies the toxin as misfolded by the ERAD pathway. Stabilization of CTA1 via glycerol inhibits the loss of its tertiary structure. This stabilization results in decreased translocation from the ER to the cytosol and increased secretion of CTA1 to the extracellular medium. Treatment with glycerol also prevents CTA1 degradation by the 20S proteasome in vitro. These data indicate that the thermal stability of CTA1 plays an important role in intoxication. These data also suggest that stabilization of CTA1 tertiary structure is a potential target for therapeutic agents. Our model asserts that CTA1 behaves as a normal ERAD substrate upon dissociation from the holotoxin. In support of this model, we have shown that the ER luminal protein HEDJ, known to be involved in ERAD, interacts with CTA1. The interactions between HEDJ and CTA1 occur only at temperatures in which the toxin is in an unfolded conformation. We have also shown that HEDJ does not affect the thermally stability of CTA1 since there is no alteration in its pattern of temperature-dependent protease sensitivity. Alteration of the normal HEDJ-CTA1 interaction via a dominant-negative HEDJ construct resulted in decreased translocation from the ER to the cytosol and, as a result, decreased intoxication. Our work demonstrated toxin resistance can result through effects on toxin structure or ERAD chaperones. To identify other potential inhibitors, we developed a novel assay to detect the activity of other AB toxins and compared it with an established toxicity assay. We generated a Vero cell line that expressed a destabilized variant of enhanced green fluorescent protein (EGFP). These cells were used to monitor the Stx-induced inhibition of protein synthesis by monitoring the loss of EGFP fluorescence from cells. We screened a panel of 13 plant compounds, and indentified grape seed extract and grape pomace extract as inhibitors of Stx activity. Grape seed extract and grape pomace extract were also shown to block the toxic activities of ETA and ricin, providing the basis for a future high-throughput screen for multi-toxin inhibitors